S-nitrosothiols, namely molecules having the basic structure R—S—N═O, where R is any organic group, e.g., S-nitrosoglutathione (GSNO), S—NO-cysteine (S—NO-Cys), S-nitroso-N-acetylpenicillamine (SNAP), and nitroso derivatives of proteins such as albumin and haemoglobin (Hb), for example SNO-albumin and poly-SNO-albumin, exert nitric oxide-like activity in vivo. They cause arterial and venous smooth muscle relaxation, inhibit platelet aggregation, and activate guanylate cyclase (Rees et al., 1989b; Rees et al., 1989a), (Rees et al., 2001), (Radomski et al., 1992). They are also involved, for example, in immunosuppression, neurotransmission, and host defence.
Vasoactive S-nitrosothiols are known to be generated in vivo (Keaney et al., 1993), (Stamler et al., 1992), (Al-Sa'doni et al., 2000). The total S-nitrosothiols in plasma has been reported to range from 40 nM to 7 μM in humans and rodents under non-inflammatory conditions (Stamler et al., 1992). S-nitrosothiol compounds can release nitric oxide when required via reactions with transition metal ions or other reducing agents. They are envisioned as a buffering system that controls intra- and extra-cellular activities of NO, and magnify the range of its action. Once formed, S-nitrosothiols can directly transfer the nitrosyl cation (NO+) to another thiol via the so-called transnitrosation reaction, which ensures the dynamic state of S-nitrosothiols in vivo (Singh et al., 1996a; Singh et al., 1996b), (Butler et al., 1997).
S-nitrosoglutathione (GSNO)
is in clinical development as a pharmaceutical composition for human or veterinary therapy or prophylaxis.
S-nitrosothiols are sensitive to air, temperature, moisture and electromagnetic radiation, and require careful storage and handling to avoid degradation (Singh et al., 1995), (Stamler et al., 2002), (Manoj et al., 2009), (Parent et al., 2013). Nevertheless, even under strictly controlled air, radiation and temperature conditions, long term storage of S-nitrosothiols, as required for pharmaceutical products, results in degradation. For pharmaceutical use, this instability poses serious problems in terms of the purity of the active agent and compliance with pharmaceutical grade requirements, as well as accuracy and predictability of the dosing of the active agent. Upon storage under inert atmosphere (argon and nitrogen) at −20° C. and in the dark, S-nitrosoglutathione, stored as a solid, has only been reported to be stable for 6 months (Parent et al., 2013). Upon storage under further controlled air, radiation and temperature conditions, S-nitrosoglutathione, prepared by a specific method said to impart stability in solid form, has only been reported to be stable for 9 months (Looker et al., 2008); (WO 2008/153762, Example 3 of the reference).
Liquid formulations of S-nitrosothiols are even more unstable and decompose quantitatively in hours, such that only about 85% of the initial S-nitrosoglutathione is present after at least 8-12 hours following storage at 4° C. to 25° C. (Looker et al., 2008); (WO 2008/153762).
Although S-nitrosothiols, for example S-nitrosoglutathione, are attractive for treating a variety of diseases, solid oral dose formulations are unsuitable, as they decompose rapidly under physiological conditions and are not able to deliver sufficient quantities of the active moiety, NO, to the desired location for extended periods of time or in a controlled manner. Parenteral injectable solutions offer a suitable form of administration and continuous infusion allows sufficient quantities of S-nitrosothiols to reach the desired location rapidly for extended periods of time and in a controlled manner. The extreme labile nature of S-nitrosothiols, such as S-nitrosoglutathione, in solution prevents formulation directly as a “ready to use liquid dosage form”. As such, the solution would need to be made up immediately (extemporaneously) from the S-nitrosothiol solid dry particles, prior to administration.
Parenteral dosage forms differ from all other dosage forms in that they must be sterile (absent of viable micro-organisms) and free from physical, chemical, and biological contaminants as they are administered by injection, infusion or implantation directly into the human or animal body. The pharmaceutical grade requirements for sterilisation of S-nitrosothiol parenteral products pose significant commercial development problems.
So-called final-filter devices for intravenous (IV) administration include filters attached to the end of the tubing of an IV administration set for removing particulates and some microorganisms such as bacteria or fungi by filtration immediately prior to entry into the IV needle for administration. The use of filters having porosities in the sub−0.5-μm region (e.g. 0.22 μm) enables, in principle, bacteria and fungi to be removed. However, the use of final-filter devices carries associated problems. Firstly, some bacteria and fungi, that are not uniformly sized or spherical, could pass through the filter membrane, as could viruses and mycoplasmas. In addition, when low concentrations of a drug are present in the solution for injection, absorption of the drug onto the filter membrane can lead to inadvertent underdosing of drug. Furthermore, fine filters when wet cannot pass air except under elevated pressure, which can lead to retention of air bubbles in the tubing during set-up and a consequent risk of injection of air to the patient during use when injection pressure is applied. The use of final-filter devices increases equipment costs, it creates a need for a constant supply of specialised consumables, and it creates a requirement for special training of regular healthcare personnel or the employment of specialists. Finally, regulatory authorities require stringent controls of this procedure and do not consider it as providing a sterile parenteral solution but only a step that would remove particulate matter.
The European Medicines Agency (EMA) has published guidelines for the selection of the most appropriate sterilisation method for a range of medicinal products. For dry powders, the preferred method according to the guidelines is sterilisation by dry heat in the sealed final container (terminal sterilisation); however, for thermosensitive medicinal products such as S-nitrosothiols, this sterilisation method would not be feasible, as the S-nitrosothiol would decompose. An alternative approach would be radiosterilisation (sterilisation by exposure to ionising electromagnetic radiation) (CPMP/QWP/054/98 corr). Sterilisation by irradiation also provides legal proprietary benefits to drug developers—the US Food and Drug Administration (FDA) categorises drugs which have been sterilised by irradiation as new drugs, meaning that an approved new drug application is required for marketing (21 CFR 310.502(a)(11)). However, S-nitrosothiols are also extremely sensitive to electromagnetic radiation, being also highly labile following free radical exposure. Indeed, an attempt to sterilise an injectable formulation of S-nitrosoglutathione within septum-sealed vials using gamma radiation was apparently unsuccessful as it was reported that an unexpected inorganic nitrate impurity was generated (Lin, et al, 2005). Furthermore, sulphur amino acids (such as cysteine in S-nitrosoglutathione) have been reported to be particularly sensitive to irradiation leading to degradation at gamma radiation levels as low as 5 kGy, possibly due to susceptibility to free radical attack (Ahn et al., 2002).
The sensitivity of S-nitrosothiols towards temperature and ionising electromagnetic radiation has hitherto prevented these methods being considered as realistic or reliable for preparing sterile, pharmaceutical grade, S-nitrosothiols. Like all methods of sterilisation, irradiation involves a compromise between inactivation of the contaminating microorganisms and damage to the product being sterilised. The imparted energy, in the form of gamma photons or electrons, does not particularly differentiate between molecules of the contaminating microorganism and those of the pharmaceutical product.
For these reasons and others, effective simple sterilisation of S-nitrosothiols such as, for example, S-nitrosoglutathione, in clinical grades of purity has been an insurmountable barrier, which has hindered the development of pharmaceutical uses of these potentially valuable drugs in human and veterinary medicine.
The present invention is based on our surprising finding that ionising radiation is effective to sterilise S-nitrosothiols with either no reduction in purity or a reduction in purity by not more than about 5.0% through degradation, so that these active agents, for example in the form of solid particles in a pre-metered dosage weight, preferably in medical vials or similar containers, can be sterilised in clinical grades of purity.